Water deficit changes the relationships between epidemiological traits of Cauliflower mosaic virus across diverse Arabidopsis thaliana accessions

Changes in plant abiotic environments may alter plant virus epidemiological traits, but how such changes actually affect their quantitative relationships is poorly understood. Here, we investigated the effects of water deficit on Cauliflower mosaic virus (CaMV) traits (virulence, accumulation, and vectored-transmission rate) in 24 natural Arabidopsis thaliana accessions grown under strictly controlled environmental conditions. CaMV virulence increased significantly in response to water deficit during vegetative growth in all A. thaliana accessions, while viral transmission by aphids and within-host accumulation were significantly altered in only a few. Under well-watered conditions, CaMV accumulation was correlated positively with CaMV transmission by aphids, while under water deficit, this relationship was reversed. Hence, under water deficit, high CaMV accumulation did not predispose to increased horizontal transmission. No other significant relationship between viral traits could be detected. Across accessions, significant relationships between climate at collection sites and viral traits were detected but require further investigation. Interactions between epidemiological traits and their alteration under abiotic stresses must be accounted for when modelling plant virus epidemiology under scenarios of climate change.


Results
The CaMV isolate Cabb B-JI successfully infected plants from all Table S2). Moreover, a highly significant variation in accession responses to CaMV infection and WD was found (Figs. 1, 2), as indicated by a significant interactive effect between accession and inoculation, and between accession and watering (Supplementary Table S3; P = 0.013 and P = 0.021, respectively). Under the WW treatment, CaMV virulence varied greatly, with some accessions exhibiting less than 11.1 ± 8.8% reduction in vegetative growth (Piq-0), while in the most CaMV-susceptible accessions vegetative growth decreased up to 60.1 ± 5.7% (Ini-0) (-38% on average across accessions; Fig. 2A). As expected from Fig. 1, the combination of viral infection and WD was even more detrimental to vegetative growth of all accessions (-58% on average across accessions; Fig. 2A).
Water deficit changes relationships between viral traits. Under WW conditions, a significant and positive correlation was found between viral accumulation and CaMV transmission rate (⍴ = 0.49, P = 0.016; Fig. 4A). Under this watering treatment, no significant rank correlation between viral accumulation and CaMV virulence could be detected (⍴ = 0.15, P = 0.481; Fig. 4B). No significant correlation between CaMV virulence and viral transmission was also found under WW treatment (⍴ = 0.13, P = 0.531; Fig. 4C). Changes in viral traits in response to WD across accessions (Fig. 2) led to an alteration of the relationships between these traits. Specifically, the correlation between viral accumulation and CaMV transmission was significantly reversed under WD (⍴ = -0.46, P = 0.024; Fig. 4D). As observed in the WW treatment, no correlation between CaMV virulence and viral accumulation (⍴ = 0.03, P = 0.872; Fig. 4E), or between virulence and viral transmission (⍴ = − 0.06, P = 0.776; Fig. 4F), was observed.

Relationships between viral traits and biogeographic origin of accessions. Correlations between
climate at the collection sites of the accessions and viral traits were investigated ( Supplementary Fig. S1). CaMV virulence was significantly positively correlated to isothermality-defined as the ratio of mean diurnal range of temperature (mean of monthly(max. temp-min. temp)) and temperature annual range-under WW, and positively, but not significantly, under WD (WW: r = 0.50, P = 0.04; WD: r = 0.27, P = 0.21; Supplementary Fig. S1). Viral accumulation under WW was significantly negatively correlated with precipitation seasonality, i.e. the coefficient of variation of monthly precipitations (WW: r = -0.43, P = 0.04; Supplementary Fig. S1). Virus accumulation under WD was significantly negatively correlated with isothermality (WD: r = -0.51, P = 0.014; Supplementary Fig. S1). Viral transmission was significantly correlated to isothermality only under WD conditions (WD: r = 0.42, P = 0.046; Fig. S1).

Discussion
In a previous independent study, we showed that water deficit altered the transmission-virulence trade-off in the CaMV-A. thaliana pathosystem 10 . However, due to the restricted number of A. thaliana accessions, we were unable to test two key assumptions of the transmission-virulence trade-off hypothesis: (1) correlation between accumulation and transmission, and (2) correlation between accumulation and virulence. Here, we measured CaMV accumulation, virulence on vegetative growth and transmission rate by the aphid M. persicae in 24 natural Iberic accessions of A. thaliana presenting a large genetic variability and grown under two contrasting watering treatments 43 . To reflect variable plant responses levels to water deficit and, potentially, to virus infection, we selected the 24 natural accessions of A. thaliana from a large variety of climatic regions and altitudes distributed evenly across the Iberian Peninsula 44 . Significant relationships between climate parameters (isothermality, temperature and precipitation patterns and seasonality) and viral traits were detected depending of the watering treatment. Climatic conditions are frequently identified as factors of local adaptation of plant species. In A. thaliana, climatic factors have been found to be significantly associated to several traits, including flowering phenology, growth and functional strategies [45][46][47] , as well as genomic regions associated with regulation of gene expression, especially at regional and micro-geographic scales 48,49 . Contrary to a study by Montes and colleagues 9 , where no relationship between Cucumber mosaic virus viral accumulation and climatic variables from the original local populations could be detected, CaMV viral accumulation under well-watered conditions was significantly negatively correlated with precipitation seasonality while under WD, a negative correlation was detected between viral accumulation and isothermality. Interestingly, vectored-transmission was positively correlated to isothermality when plants were submitted to a WD. Under WD, CaMV accumulation within low temperature fluctuations environment-accessions was reduced while vectored-transmission would be higher than in other accessions. These relationships may result from the interrelationships between local climate conditions, occurrence of selective pathogens and the functional strategies of the plant genotypes 9,20 . The adaptive value of these relationships will require further investigations combining experiments under field and controlled conditions. Plant signaling pathways and responses to various abiotic stresses partly overlap those induced by viral infection, and their mutual interference is not a novel concept. Indeed, the effect of abiotic plant stresses on viral accumulation and virulence through the hijacking of plant signaling and defense pathways has received recent attention 21,22,50 . In accordance with previous results 20 , we showed here that application of a water deficit to CaMV-infected A. thaliana leads to higher virulence, and was overall more detrimental to plant performance compared with virulence under well-watered condition regardless of accession. Under WD, CaMV accumulation was altered in 10 accessions; in most cases there was a significant increase of this viral trait. While a negative (or no) effect of WD on viral accumulation has already been shown in several viral pathosystems 10,25,31,32 , to our knowledge, such a positive effect on virus accumulation has not previously been reported.   51 ]. WD applied to CaMV-source plants did not significantly impact CaMV transmission rate, except in four accessions. In these four accessions, WD had a contrasted effect on the transmission success, linked to the accession origin. While CaMV transmission by the aphid M. persicae increased in Can-0 and Ini-0 accessions under water deficit, it decreased in Bos-0 and Lam-0 accessions.
A positive correlation between CaMV transmission rate and accumulation was observed, which had not been detected when using fewer accessions 10 . This discrepancy might be explained by the different growing conditions (8-h vs. 12-h day length) used in the two studies. The positive correlation between CaMV transmission rate and accumulation observed here, and already shown in other pathosystems, might reflect the fact that a high availability of virus particles within plant cells increases the chance of acquisition by the vector 37,52 . However, in this study, factors other than accumulation might also explain alteration of virus transmission when plants experienced a water deficit. Indeed, in the specific case of Can-0 accession, which exhibited a significant increase of CaMV transmission under WD, accumulation did not seem to be the limiting factor as virus accumulation remained stable whatever the watering treatment. This observation was supported by the inverse relationship between CaMV transmission and virus accumulation under WD. As a result, plants with a lower virus content became significantly better source plants for vectored-transmission under WD. A lack of positive correlation between accumulation and transmission rate has already been shown in CaMV-infected B. rapa source plants experiencing severe WD 31 . The significant increase in transmission rate was suggested to be due to a change in host plant physiological status that could trigger a direct effect on virus behavior 53,54 . As a consequence, these rapid changes in virus behavior may actually predisposes the infected plant to a more efficient virus acquisition and transmission by aphid vectors 53,54 . This remarkable phenomenon has been termed 'transmission activation' and can be triggered by abiotic stresses such as CO 2 treatment 55 .
We were unable to find any other relationship between viral traits. Unsuccessful validation of the trade-off hypothesis-i.e. a negative correlation between virulence and transmission-might be explained by the fact that one of the two required assumptions of this trade-off-i.e. a positive correlation between accumulation and virulence-was not demonstrated in our model system. Moreover, the trade-off hypothesis is an adaptive hypothesis that supposes a common evolutionary history between the host and the pathogen, suggesting efficient development of the parasite without harming the host 35 . Indeed, with the rise of metagenomics, sequence data collected in natura confirm that most virus-infected plants are asymptomatic 56 . Also, it is important to note that, in our system, the virus isolate did not co-evolve with these specific plant accessions even though several other CaMV isolates are reported to infect natural A. thaliana populations in Spain 41 . The isolate CaMV Cabb B-JI was originally isolated from B. rapa and fixed by cloning the genome sequence 57 . Moreover, the trade-off hypothesis, which assumes a simplified biology where virulence and transmission of the parasite are independent of the characteristics of the host, remains controversial 58 . In fact, viral traits are the result of multiple interactions within the host, such as immune responses to counteract the development of infection.
In conclusion, our results reaffirm that water deficit might have substantial effects on key viral traits, with epidemiological consequences for plant viral disease. The multi-faceted relationships between virulence, viral accumulation and vectored-transmission according to the environmental conditions experienced by the host invite further investigation. Experiments were conducted in the PHENOPSIS facility. This phenotyping platform allows automated watering, weighing and imaging of 504 potted plants under strictly controlled environmental conditions 42 . Three to five seeds were sown at the soil surface in 225-ml pots filled with a 30:70 (v/v) mixture of clay and organic compost (substrate SP 15% KLASMANN) and placed randomly in the PHENOPSIS growth chamber. Soil water content was estimated for each pot before sowing, as previously described 42 . The soil surface was moistened with deionized water, and pots were placed in the dark for 2 days at 12 °C air temperature and 70% air relative humidity. Pots were dampened with sprayed deionized water three times a day until germination. After the germination phase (ca. 7 days), plants were cultivated under 12-h day length at 200 μmol m -2 s -1 photosynthetic photon flux density at plant height. Air temperature was set to 20 °C, and air relative humidity was adjusted in order to maintain constant water vapor pressure deficit at 0.6 kPa. At the appearance of the cotyledons, one plant was kept per pot, and the temperature was set at 21/18 °C day/night, while the vapor pressure deficit was set at 0.75 kPa. Each pot was weighed daily and watered with deionized water to reach the target soil relative water content. Soil relative water content was maintained at 1.4 g H 2 O g -1 dry soil (WW) until application of the treatments. CaMV-or mock-inoculation (see below) was performed at the emergence of the tenth rosette leaf. WD was applied 1 week after inoculation-the approximate timing of first symptom appearance. Irrigation of half of the CaMV-and mock-inoculated plants was stopped to reach WD treatment at 0.50 H 2 O g -1 dry soil, reached after 7 days of water deprivation, and then maintained at this value until the end of the experiment. Under WW, soil relative water content was maintained at 1.4 g H 2 O g -1 dry soil. All environmental data, including daily soil water content, air temperature, and vapor pressure deficit, are available in the PHENOPSIS database 59 . non-circulative aphid-transmitted virus-was used in this study 57 . Virus particles were purified from CaMVinfected Brassica rapa cv. "Just Right" (turnip) plants as previously described 60 . The quality and quantity of purified virus were assessed by polyacrylamide gel electrophoresis under denaturing treatments (12% SDS-PAGE) and by spectrometric measurements at 230, 260, and 280 nm (NanoDrop 2000 spectrophotometer). Virus concentration was estimated by spectrometry using the formula described by Hull and Shepherd 60 . At the 10-leaf stage, A. thaliana source plants were mechanically inoculated as previously described 10 . Briefly, CaMV-infected turnip extract was prepared from 1 g of infected leaf material [turnip leaves presenting systemic symptoms collected at 21 days post inoculation (dpi)] ground in 1 mL of distilled water with carborundum. Purified CaMV particles were then added to this mix at a final concentration of 0.2 mg mL -1 to optimize infection success. For each inoculated plant, 10 μL of the solution described above was deposited on each of three middle-rank leaves. Leaves were then rubbed with an abrasive pestle. The control group was mock-inoculated in a similar way to mimic the wounds induced by mechanical inoculation. Mock-inoculation was performed with a mix containing non-infected turnip plant extract and the buffer used for virus purification (100 mM Tris-HCl, 2.5 mM MgCl 2 , pH 7). All plants were randomly inoculated, independently of accession and watering regime.

Measurement of plant traits.
Harvests were carried out at 30 dpi following transmission experiments (see below). Each rosette was cut, fresh mass was measured then the tissue was kept in deionized water for 24 h at 4 °C to determine the water-saturated weight (mg). Collected rosettes were subsequently oven-dried at 65 °C for at least 5 days, and their dry masses determined. Virulence, described as the impact of CaMV infection on vegetative growth 61  Transmission assays. CaMV transmission efficiency was assessed at 30 dpi. Batches of 20 M. persicae larvae (L2-L4 instars) were starved for 1 h before being transferred to the rosette center of a CaMV-mechanically infected source plant for virus acquisition; 11 symptomatic mechanically infected source plants were used per accession and watering treatment. When aphids stopped walking and inserted their stylets into the leaf surface, they were allowed to feed for a short 2-min period. Viruliferous aphids were then immediately collected in a Petri dish and individually transferred to 1-month-old Col-0 plantlets (receptor plants) grown under nonstressing treatments (one aphid per receptor plant; nine receptor plants per source plant) as described 10 . After an inoculation period of 3 h, aphids were eliminated by insecticide spray (0.2% Pirimor G). Receptor plants were then placed in a growth chamber with the same treatments of air humidity, temperature and light as source plants and maintained under non-stressing conditions. Symptoms of virus infection were recorded 21 days later by visual inspection on receptor plants, following the procedure previously reported 10 and virus transmission rate was then calculated. Following transmission experiments, three leaf discs in the center of the rosette were randomly collected on each mechanically infected source plant and stored at -80 °C for further nucleic acid extraction and virus quantification.
Plant DNA extraction. Total DNA from CaMV-infected samples (pool of three leaf discs collected per plant) was extracted according to a modified Edwards' protocol with an additional washing step with 70% ethanol 62 . DNA was resuspended in 50 μL of distilled water, and ten-fold dilutions were used as qPCR templates. Quality and quantity of the extracted total nucleic acid were assessed by spectroscopic measurements at 230, 260 and 280 nm (NanoDrop 2000 spectrophotometer). www.nature.com/scientificreports/ Data analyses. All analyzes were performed in the programming environment R 64 . Variations of vegetative growth (aboveground dry mass), viral accumulation and transmission rate among accessions in response to virus infection and watering treatment were analyzed in both parametric (type III) and non-parametric (rankbased) ANOVAs. Non-parametric procedure was used because of unbalanced sampling across factor levels and risks of deviation from Normality and heteroscedasticity. Data of the two experiments were analyzed together since no significant interactive or main effect of experiment was found for aboveground dry mass, viral accumulation, and viral transmission of the control accession Col-0 (Supplementary Table S7). For each accession, the effects of the treatments on traits were analysed by non-parametric tests for two (Wilcoxon) or more (Kruskal-Wallis) samples. For each accession, relative change was calculated as the ratio of the difference between the trait value of each replicate plant and mean trait value under control conditions (mock:well-watered) to the mean trait value under control conditions. Non-parametric ANOVAs were performed using the raov function of the Rfit package 65 . Bootstrapped 95% confidence intervals (CI) of mean trait values were computed following the mean_cl_boot procedure of the Hmisc package 66 . We used the R package cvequality (Version 0.1.3) 67 to test for significant differences between coefficients of variation. Nonlinear models were fitted using the nls function and 95% confidence intervals for the parameters of fitted models were computed with the confint function of the package mass 68 . The effect of watering on transmission rate was analyzed using generalized linear models with the binomial model in the glm function of the stat package applied to the proportion of infected and uninfected plants in the transmission assays. Relationships between traits were examined with Spearman's rank-order coefficients of correlation (⍴) using the function cor.test. Relationships between traits and climate at the collection sites of the accessions (obtained from WorldClim v. 1.4, http:// world clim. org) 69 were examined in generalised least squares models in order to take spatial autocorrelation into account. First, we tested the bivariate relationships between traits and climatic variables, then we added a Gaussian autocorrelation structure to the model. Pearson's correlation coefficients are reported with GLS P values.
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